Cleavage of Nucleic Acid
Many techniques of molecular biology depend on the ability to cleave DNA or RNA molecules specifically at defined locations. Restriction enzymes cleave double-stranded DNA at specific sequences that are usually palindromic and 4-6 base pairs in length. Several hundred restriction enzymes have been discovered, most of which only cleave double-stranded DNA molecules. Several restriction enzymes have been shown to have the ability to cleave single-stranded DNA, albeit with reduced efficiency, at sites that bear the sequence that is normally recognized in double-stranded DNA. While the large number of different restriction enzymes cleave double-stranded DNA at a variety of sites, these enzymes cleave only at sites whose sequences conform to the substrate sequence specificity of the enzyme, and do not cleave at all points that might be desired by the investigator. Therefore, restriction enzymes limit a cleavage reaction to both a specific nucleotide sequence and to the use of double-stranded DNA.
Class IIs restriction enzymes, such as Fok 1, cleave DNA at a site other than their recognition sequences. Fok I can be directed to cleave single-stranded DNA at selected sites through the use of adaptor oligonucleotides that direct it to the DNA (Podhajska et al., Gene, 40, 175-182; 1985). The adaptor must contain two regions--one that serves as the recognition site for the enzyme and another that hybridizes to the single-stranded DNA. The specificity of the binding of the adaptor to its target may be relatively low due to the ability of the enzyme to tolerate mismatched base pairs and the need to incubate the reaction at temperatures below 40.degree. C. Only DNA has been shown to be a substrate for cleavage in this system.
Ribozymes, RNA molecules that possess self-catalytic activity, can be targeted to cleave nucleic acid. However, the specific target cleavage sites have a sequence requirement (Symons, R. Ann. Rev. of Biochem., 61:641-671, 1992).
Other methods of cleaving nucleic acids include the use of non-specific nucleases. A nuclease is an enzyme that cleaves nucleic acids. Endonucleases, such as the restriction endonucleases discussed above, cleave nucleic acids by hydrolysis of internal phosphodiester bonds. Although restriction endonucleases cleave only at specific nucleotide sequences, other endonucleases, such as Mung Bean nuclease, are not sequence-specific. In contrast, exonucleases cleave nucleic acid chains from the ends. An example of a structure-specific nuclease is snake venom phosphodiesterase I which is a nuclease that degrades single-stranded nucleic acids. Non-sequence-specific nucleases, either exonucleases or endonucleases, cannot he used directly to cleave nucleic acid molecules in a sequence specific manner.
RNA can be cleaved at specific sites through hybridization of adaptor molecules that serve as sequence-specific recognition sites for RNases such as RNaseP. (Li, et al., Proc. Natl. Acad. Sci., 89:3185-3189, 1992.)
There is a need in the art of molecular biology techniques for a method to cleave nucleic acids at any specific sequence that is not limited to sequences recognized by restriction endonucleases.
DNA Polymerase
DNA polymerases (DNAPs) catalyze the synthesis of a DNA chain. Additionally, many DNAPs are known to have nuclease activity.
Some DNAPs are known to remove nucleotides from the 5' and 3' ends of DNA chains (Kornberg, et al., DNA Replication, 2d ed., W. H. Freeman and Co., publishers, 1992). These activities are usually termed "5' exonuclease" and "3' exonuclease", respectively. For example, the 5' exonuclease activity located in the N-terminal domain of several DNAPs participates in removal of RNA primers during lagging strand synthesis during replication and the removal of damaged nucleotides during repair. Some DNAPs, such as one isolated from E. coli (DNAPEcl), also have a 3' exonuclease activity responsible for proof-reading during synthesis (Kornberg, supra).
A DNAP isolated from Thermus aquaticus, called Taq DNA polymerase (DNAPTaq), has a 5' exonuclease activity, but lacks a functional 3' exonucleolytic domain (Lawyer, et al., J. Biol. Chem. Sci., 12:288, 1987). Derivatives of DNAPEcl and DNAPTaq, respectively called the Klenow (DNAPKln) and Stoffel (DNAPStf) fragments, lack 5' exonuclease domains as a result of enzymatic or genetic manipulations (Brutlag, et al., Biochem. Biophys. Res. Commun., 37:982, 1969; Erlich, et al., Science, 252:1643, 1991; Setlow, et al., J. Biol. Chem., 247:232, 1972). The 5' exonuclease activity of DNAPTaq is reported to require concurrent synthesis (D. H. Gelfand, PCR Technology; Principles and Applications For DNA Amplification, Henry A. Erlich, ed. Stockton Press, 17, 1989). Although mononucleotides predominate among the digestion products of the 5' exonucleases of DNAPTaq and DNAPEcl, short oligonucleotides (.ltoreq.12 nucleotides) can also be observed, implying that these so-called 5' exonucleases can function endonucleolytically (Setlow, supra; Holland, et al. Proc. Natl. Acad. Sci. USA., 88:7276, 1991). Thus, we prefer to call these activities "5' nucleases".